Over the past several decades methods have been devised for constructing genetic elements effective to code for virtually any desired gene product; and for preparing antisense oligos and related substances effective for modifying the expression of virtually any gene; and for preparing peptides and proteins effective for directly acting within cells. A major limitation in utilizing such new technologies for a host of valuable research and therapeutic applications has been the difficulty of efficiently delivering large polar substances into the cytosol of animal cells without incurring undue damage to those cells. Thus, a simple, safe, and effective means for delivering large polar substances into animal cells has many immediate and valuable applications in biological and medical research, and may also have a wide range of therapeutic applications in the near future.
While a number of delivery systems are currently available for delivering large polar substances into the cytosol of cells, most of these delivery systems suffer from at least one, and usually several of the following limitations: substantial toxicity to the recipient cells; low efficiency, particularly in the presence of even a few percent of serum; undue complexity of use; and, poor reliability and reproducibility. Such delivery systems include ones developed by the applicant: scrape delivery (Partridge, 1996); weak-acid transport engines (Summerton, 2000); osmotic delivery (Morcos, 2001); and, mixed-base polyamines (Morcos, 2001). In addition, many other types of delivery systems have also been developed, including: microinjection (Chin, 1990); multiple types of liposomes (Thierry, 1992); streptolysin O (Spiller, 1995); electroporation (Bergan, 1996); strong-base polyamines (Boussif, 1995); multiple natural and designed strong-base peptides (Lemaitre, 1987; Derossi, 1994); and, natural and designed weak-acid peptides (Pichon, 1997).
Further, a number of viral and excreted cellular proteins are known to penetrate animal cell membranes, often by virtue of a short strong-base (polycationic at neutral pH) amphiphilic segment of the protein, typically with a segment length of around 12 to 18 amino acids. Such sequences are commonly referred to as “protein transduction domains”. Representative well-studied examples include a short segment of tat protein of the HIV virus (Mann, 1991), a short segment of the VP22 protein of Herpes virus (Elliott, 1997), and a short segment of an excreted neural protein of the fruit fly (Derossi, 1994).
Synthetic versions and close analogs of these membrane penetrating peptide sequences have been developed and used to deliver a variety of substances into animal cells. Such membrane penetrating peptides typically contain a lipophilic face or end, plus a face or end containing multiple strong-base amino acids selected from arginine and lysine. This peptide structural type consisting of both a lipophilic region and a strong-base region has been further developed by Prochiantz and coworkers (described in PCT published Patent application WO 97/12912) to give a designed peptide of tryptophans and arginines—affording a peptide with a membrane penetrating capacity substantially greater than natural peptide sequences reported to date. Applicant has designed and developed a different series of strong-base peptides composed of leucines and lysines, and cell testing results indicate that such peptides have a still greater membrane penetrating capacity (unpublished results).
Many of these systems, particularly the natural and designed strong-base peptides, are reasonably effective for delivering large polar substances into the cytosol of cells. However, they are typically of limited utility because of their toxicity to cells. To a large extent this appears to be because their delivery mechanism entails permeabilizing the plasma membrane, which allows loss from the cells of salts, sugars, vitamins, amino acids, nucleotides, and a host of other essential cellular constituents.
It has been postulated that such toxicity could be largely avoided by developing delivery systems which utilize an indirect endocytosis-mediated delivery mechanism wherein the plasma membrane is never permeabilized.
In an effort to reduce the toxic effects commonly incurred with direct-entry delivery systems, several mixed-base delivery systems have been developed with the goal of achieving indirect delivery via endocytosis. One representative mixed-base system uses polylysine wherein the lysine side chains are acylated with histidines (Pichon, 2000). This gives a polymeric composition wherein the strong-base alpha-amino moieties of the histidines serve to bind electrostatically to the negatively charged surface of the plasma membrane of cells. Subsequent endocytosis and acidification within the endosome serves to ionize the weak-base imidazole moieties of the histidines giving a composite charge density on the polymeric composition sufficient to permeabilize the endosomal membrane. This allows co-endocytosed substances (cargo) to pass from the endosome into the cytosol of the cell.
Another mixed-base delivery system is marketed by GENE TOOLS, LLC. It utilizes ethoxylated polyethyleneimine (EPEI) for delivering Morpholino antisense oligos into the cytosol of cultured cells (Morcos, 2001). EPEI contains in roughly equal portions both moderately strong base moieties having pKa values above pH 7, and weaker-base moieties having pKa values in the range of about 5.5 to 7. In this delivery system the higher-pKa base moieties of EPEI (ionized at neutral pH) serve to bind electrostatically to the negatively charged surface of the plasma membrane of cells. Subsequent endocytosis and acidification within the endosome serves to ionize the weaker-base moieties, resulting in a composite charge density on the EPEI sufficient to permeabilize the endosomal membrane, thereby allowing co-endocytosed cargo to pass from the endosome through the permeabilized endosomal membrane into the cytosol of the cell.
While these mixed-base delivery systems are indeed less toxic to cells than the strong-base delivery systems, nonetheless, they are still somewhat toxic. In addition, their efficiency is much reduced in the presence of just a few percent of serum. This poor activity in the presence of serum, a problem shared with most other delivery systems, is a significant limitation because numerous cell types, particularly primaries, are damaged in the absence of serum.
The good efficacy with reduced toxicity seen with mixed-base delivery systems suggested the possibility that simply deleting most or all of the strong-base moieties (cationic at neutral pH) while keeping the weak-base moieties (non-ionic at neutral pH) might alleviate the cell toxicity problem. A paper in the scientific literature indicates that something along this line has already been reported (P. Midoux, et al: Membrane permeabilization and efficient gene transfer by a peptide containing several histidines. Bioconjugate Chemistry 9: 260, 1998). Initially other workers had identified an anionic peptide from the N-terminal segment of the HA-2 subunit of the influenza virus hemaglutinin which is involved in fusion of the viral envelope with the endosomal membrane upon acidification of the endosome (Plank, 1994). That peptide sequence was subsequently modified slightly to increase its acid-triggered membrane permeabilization properties (Murata, 1991). Thereafter, the modified sequence was further modified by replacing its anionic moieties with cationic lysines to give a strong-base version of the originally-polyanionic peptide (Murata, 1992). While this strong-base version was effective for permeabilizing cell membranes, albeit with significant toxicity, that permeabilizing activity was largely lost in the presence of serum.
With the objective of decreasing toxicity and achieving activity in the presence of serum, Midoux and co-workers subsequently replaced the 5 lysines of the peptide with 5 histidines (Midoux, 1998), to give a peptide with four amino acid types (5 weak-base histidines, 8 non-ionic hydrophilic amino acids, 5 aliphatic lipophilic amino acids, and 5 aromatic lipophilic amino acids). Since glycines disfavor the alpha helical conformation, because of this peptide's high and well dispersed glycine content, this peptide most likely exists in a largely unstructured random coil in aqueous solution. FIG. 13a of the instant application shows the sequence of this peptide (prior art).
It is also noteworthy that in this semi-natural peptide developed by Midoux only a minority (44%) of the side chains appear optimal for membrane binding and subsequent pH-triggered permeabilization. More specifically, only 22% (the L and I side chains) appear optimal for membrane binding, and only 22% (the H side chains) are expected to contribute to membrane permeabilization upon acidification within the endosome. It seems likely that the remaining majority of the side chains (56%) of this peptide may have served in the original natural peptide sequence to properly integrate that sequence into the rest of the protein in which it was an integral part.
In sharp contrast to the case for the semi-natural unstructured weak-base peptide of Midoux, the designed peptides of the instant invention, as illustrated in FIG. 13b, have an amino acid composition that virtually assures a regular alpha helical conformation at neutral pH. In that alpha helical conformation the peptide has two precisely delineated faces, a lipophilic face and a weak-base face. Most or all side chains of the lipophilic face serve explicitly for membrane binding at neutral pH, and most or all side chains of the weak-base face contribute to membrane permeabilization upon acidification within the endosome. Thus, in these designed highly structured delivery peptides of the instant invention most or all of the side chains are designed to be both optimal for and suitably positioned to carry out the two key functions required for an indirect endocytosis-mediated cytosolic delivery process, those two functions being: a) membrane binding by the lipophilic face at neutral pH; and, b) membrane permeabilization by the weak-base face at acidic pH.
While the weak-base peptide of Midoux does achieve delivery of cargos into cultured cells, even in the presence of serum, the experience of Prochiantz in designing from scratch a highly effective tryptophan/arginine peptide and my experience in designing from scratch an even more effective leucine/lysine peptide (unpublished results) led me to speculate that it might be possible to substantially improve delivery efficiency, reduce toxicity, and achieve better activity in the presence of serum by creating from scratch a delivery agent expressly designed and optimized for indirect endocytosis-mediated cytosolic delivery.